Host-microbiota interactions have been implicated in diverse effects ranging from immune system development to metabolism and behavior. Recent studies implicate a pivotal role for microbial metabolites that may act on the host. In the most recent funding period of our grant, we have shown that in the simple animal host Caenorhabditis elegans, chemosensory recognition of specific virulence-associated secondary metabolites produced by Pseudomonas aeruginosa activates a G protein-dependent signaling pathway that alters the neuronal expression pattern of DAF-7, a neuronal TGF? ligand with restricted expression that has previously been shown to regulate diverse aspects of C. elegans physiology. Induction of DAF-7 expression in the ASJ sensory neuron pair acts on specific promoters to promote behavioral avoidance by C. elegans. This behavioral phenotype and quantitative analysis of DAF-7 expression changes is further complemented by calcium imaging with GCaMP of ASJ neuron activation in response to bacterial metabolites. Our data have established an experimental system in a simple, genetically tractable host with a well-defined nervous system, which we can use to define the molecular mechanisms by which microbial metabolites can influence host physiology and behavior. From the analysis of over twenty mutants that exhibit defective daf-7 transcriptional responses in the ASJ neurons in response to P. aeruginosa, we have identified distinct candidate calcium-dependent signaling pathways that regulate the host neuroendocrine response to P. aeruginosa metabolites. We have also carried out preliminary RNA-seq of ASJ neurons that have been separated and FACS-sorted, which reveal a number of candidate genes involved in the regulation of C. elegans neuronal responses to P. aeruginosa. We will conduct molecular genetic analysis to establish the signaling pathways that transduce the detection of microbial metabolites to altered DAF-7 transcription, and we will define the mechanisms by which DAF-7 expression in the ASJ neurons in regulated. We will also test the hypothesis that microbial metabolites also modulate neuroendocrine insulin signaling, and carry out a systematic functional analysis of the transcriptional responses of the ASJ neuron pair to microbial metabolites. We anticipate that defining the molecular mechanisms underlying how specific microbial metabolites can modulate the activities of conserved neuroendocrine signaling in C. elegans will yield insights into evolutionarily conserved interactions between microbiota and their animal hosts.